Multilayer gas-permeable container for the culture of adherent and non-adherent cells

ABSTRACT

A multi-layer, flexible, gas-permeable film ( 10 ) suitable for forming a cell culture container ( 20 ), the film ( 10 ) comprising a first layer ( 12 ) composed of a polystyrene having a thickness within the range of 0.0001 inches to about 0.0010 inches and, a second layer ( 14 ) adhered to the first layer ( 12 ) composed of a polyolefin having a thickness within the range of 0.004 inches to about 0.015 inches.

TECHNICAL FIELD

This invention relates to multi-layer films, and containers formedtherefrom for the in vitro culture of cells. Specifically, the inventionis directed to a multi-layer, flexible, gas permeable container havingan inner growing surface of polystyrene, which is conducive to theculture of cells.

BACKGROUND ART

There are two major types of cells grown in vitro: suspension cells(anchorage-independent cells); and adherent cells (anchorage-dependentcells). Suspension or anchorage-independent cells can multiply, invitro, without being attached to a surface. In contrast, adherent cellsrequire attachment to a surface in order to grow in vitro. Additionally,some non-adherent cells grow best on a surface that promotes adherentcell growth.

It is known to grow adherent cells, in vitro, in polystyrene flasks.Polystyrene is the most common type of plastic used in the manufactureof rigid, gas impermeable cell culture flasks or plates. It is thoughtthat polystyrene promotes the growth of adherent cells because of itsability to maintain electrostatic charges on its surface which attractoppositely charged proteins on the cell surfaces. However, to date, theavailable polystyrene culture containers have been of the rigid flask orplate type because polystyrene is known in the art as a rigid,gas-impermeable plastic.

Cells are commonly cultured in a growth medium within polystyrene orother containers placed in enclosed incubators. In addition to providinga certain degree of isolation from pathogens, the incubators maintain aconstant temperature, usually 37° C., and a constant gas mixture. Thegas mixture must be optimized for a given cell type, and be controlledfor at least two parameters: (1) partial pressure of oxygen (pO₂) toserve the aerobic needs of the cells, and (2) partial pressure of carbondioxide (pCO₂) to maintain the pH of the growth medium. Since the knowntypes of rigid cell culture containers are gas impermeable, their lidsor caps are not sealed onto the containers. Rather, they are offsetsufficiently to allow gas exchange through a gap or vent between the capand the container. Such a container is disadvantageous for clinical usesbecause the vent might allow contamination of the culture or lead toaccidents involving biohazardous agents.

In addition to polystyrene flasks, others have constructed flexible,breathable containers for containing adherent cells to be grown invitro. For example, the commonly assigned U.S. Pat. No. 4,939,151provides a gas-permeable bag with at least one access port. This allowsfor a closed system (ie., one without a vent). The bag disclosed in the'151 Patent is constructed from two side walls. The first side wall ismade of ethylene-vinyl acetate (“EVA”) which may be positively ornegatively charged. The second side wall is constructed from a gaspermeable film such as ethylene-vinyl acetate or a polyolefin. The firstside wall is sealed to the second side wall along their edges. While EVAcan hold an electrostatic charge, the charge has the undesirabletendency to decay over time. Eventually, the decay will render thecontainer ineffective for growing adherent cells.

It has been found that the cell growth rate within a sealed containermay be influenced by the gas permeability characteristics of thecontainer walls. The optimal gas requirements, however, vary by celltype and over the culture period. Thus, it is desirable to be able toadjust the gas permeability of the container. The polystyrene flask, andthe flexible flask which is entirely constructed from a monofilm, do notprovide for such adjustability.

SUMMARY OF THE INVENTION AND OBJECTS

The present invention provides a multi-layer, co-extruded film suitablefor producing gas-permeable cell culture bags. The film has anultra-thin first layer of polystyrene having a thickness from about0.0001 inches to about 0.0010 inches. The film has second layer adheredto the first layer made of a polyolefin. The polyolefin acts as aflexible substrate for the polystyrene to provide a flexible, gaspermeable film. Thus, the second layer is sometimes referred to as thesubstrate layer.

The film may also have an adhesive tie layer interposed between thefirst and second layers. The film may also have one or more additionalouter layers of polyolefin (such as polypropylene) to provide strengthand scratch resistance, as well as additional tie layers interposedbetween these additional layers.

The film most preferably has the following physical characteristics: (1)a mechanical modulus of between about 10,000 and 30,000 psi (ASTM D790); (2) an oxygen permeability within the range of about 9-15 Barrers;(3) a carbon dioxide permeability of 40-80 Barrers; (4) a nitrogenpermeability of 10-100 Barrers, and (5) a water vapor transmission rateof not more than 20 (g mil/100 in²/day). Preferably, the film shouldhave an optical clarity of between about 0.1% to about 10% as measuredby a Hazometer in accordance with ASTM D1003.

The present invention also provides a flexible, gas-permeable cellculture container constructed from the above described films, with thepolystyrene layer forming the inner surface of the container.

Another aspect of the present invention provides a flexible,gas-permeable cell culture container whose gas permeability may beadjusted to best match the requirements of the cell being cultured inthe container. The multi-layer structure of the present film allows oneto vary the material of the second layer or substrate layer and itsthickness to achieve the desired or predetermined gas permeabilityrequirements for cell growth. Preferably, the type and thickness of thesubstrate layer will be selected to optimize cell growth.

Another aspect of the invention provides for various embodiments ofculture containers some of which are advantageous for growing adherentcells, non-adherent cells, and both.

Another aspect of the invention is to provide a flexible, gas permeablecell culture container having a first side that is suitable for growingadherent cells, a second side for growing non-adherent cells, andindicia on the container for indicating the first side from the secondside.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of a two-layer, gas-permeable, flexiblefilm of the invention;

FIG. 2 is a cross-sectional view of the flexible, gas-permeable cellculture container of the invention;

FIG. 3 is a cross-sectional view of a three-layer, gas-permeable,flexible film of the invention;

FIG. 4 is a cross-sectional view of a four-layer, gas-permeable,flexible film of the invention;

FIG. 5 is a cross-sectional view of a five-layer, gas-permeable,flexible film of the invention;

FIG. 6 is a cross-sectional view of a flexible, gas permeable containerfor the growth of non-adherent cells;

FIG. 7 is a cross-sectional view of a flexible, gas permeable containerfor the growth of both adherent and non-adherent cells;

FIG. 7a is a plan view of the container of FIG. 7 showing a geometricindica to distinguish the adherent side from the non-adherent side;

FIG. 8 is a cross-sectional view of a flexible, gas permeable containerfor the growth of non-adherent cells having a clear panel for inspectionof the contents; and,

FIG. 9 is a plan view of a flexible, gas permeable container of thepresent invention having access ports.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a multi-layer, gas-permeable flexiblefilm, having a surface layer formed of polystyrene, and a cell culturecontainer constructed therefrom, having an inner surface of polystyrene.

I. The Film Components

FIG. 1 shows a two-layer film 10, of which the first layer 12 of thefilm forms an inner cell growth surface when fabricated into a cellculture container 20 (FIG. 2). The film 10 having an effective thicknessto allow cell culture growth. The first layer 12 is an ultra-thin layerof polystyrene, preferably having a thickness from about 0.0001 inchesto about 0.0010 inches, more preferably 0.0002 inches to about 0.0006inches, and most preferably about 0.0003 inches. The polystyrenematerial may be selected from, but not limited to, polystyrenes such ashigh impact polystyrenes (“HIPS”) which are a general purposepolystyrene modified by polybutadiene rubber. Such a polystyrene is soldby Dow Chemical Company under the product designation Styron 47827,Natural.

The second layer 14 is composed of a polyolefin. Preferably, thepolyolefin includes a polymer alloy comprising three components:styrene-ethylene-butadiene-styrene (“SEBS”) block copolymer (40%-85% byweight), ethylene vinyl acetate (0-40% by weight), and polypropylene(10%-40% by weight) as described in the commonly assigned U.S. Pat. No.4,140,162 which is incorporated herein by reference. Such a polymeralloy is sold by Baxter International Inc. under the product designationPL-732®.

It is also desirable to use, for the second layer 14, other three andfour component polymer alloys such as those disclosed in co-pending, andcommonly assigned patent application Ser. No. 08/153,823 which isincorporated herein by reference. One such group of polymer alloysconsists of a first component of a polypropylene which constitutesapproximately 30-60% by weight of the polymer alloy. The secondcomponent is preferably an ultra low density polyethylene orpolybutene-1 which constitute approximately 25-50% by weight of thepolymer alloy. The third component of the polymer alloy is preferably adimer fatty acid polyamide (which should be interpreted to include theirhydrogenated derivatives as well), which constitutes approximately 5-40%by weight of the polymer alloy. The fourth component is acompatibilizing polymer that may be selected from various blockcopolymers of styrene with dienes or alpha olefins; the compatibilizingpolymers may be modified with minor amounts of chemically activefunctionalities such as maleic anhydride. For example, thecompatibilizing polymer may be an SEBS block copolymers. The fourthcomponent should constitute between 5-40% by weight of the polymeralloy.

Preferably, the second layer 14, (which may sometimes be referred to asthe substrate layer 14) has a thickness within a range of about 0.004inches to about 0.015 inches, more preferably 0.005 inches to about0.012 inches, and most preferably 0.006 inches to about 0.008 inches.

In another embodiment of the invention (FIG. 3), the film may include afirst tie layer 16 interposed between the first and second layers 12 and14. Preferably the first tie layer is a gas permeable olefin and morepreferably an ethylene polymer containing vinyl acetate within the rangeof 16%-30% by weight and most preferably ethylene vinyl acetate with 28%vinyl acetate. Other examples of these polymers include those sold byQuantum Chemicals under the trade name Bynel. It is also possible to useSEBS block copolymers as the first tie layer such as those sold by ShellChemical Company under the tradename Kraton.

The first tie layer 16 adheres the first layer 12 to the second layer14. The first tie layer 16 has a thickness preferably within a rangefrom about 0.0002 inches to about 0.0012 inches, more preferably fromabout 0.0004 inches to about 0.0010 inches, and most preferably about0.0005 inches.

In yet another embodiment of the invention (FIG. 4), the film shown inFIG. 3 may also have a skin layer 18 adhered to the second layer 14opposite the first layer 12, to form an outer skin which adds strengthand scratch resistance to the film 10. The skin layer 18 is preferablyformed from homo and copolymers of polypropylene, more preferablypolypropylene polymers modified by rubber. Such polymers would includethose sold by Mitsui under the trade name Admer™. The skin layer 18preferably has a thickness within a range of from about 0.0001 inches toabout 0.002 inches, and more preferably 0.0005 inches. The skin layercould also be composed of a polyethylene.

FIG. 5 shows the film of FIG. 4 except with a second tie layer 19interposed between the skin layer 18 and the substrate layer 14 toadhere the skin layer 18 to the substrate layer 14. The second tie layer19 may be composed of similar components as identified for the first tielayer such as modified polyethylenes.

II. Construction of the Film and its Physical Characteristics

In forming the film 10 of FIG. 1, the ultra-thin layer of polystyrene 12is co-extruded on the substrate layer 14 using a typical feedblockco-extrusion method.

The resultant film 10 should have a flexural modulus preferably withinthe range of 5,000-300,000 psi, more preferably within the range of10,000-200,000 psi, and most preferably 10,000-30,000 psi as measured inaccordance with ASTM D 790. The film should have an oxygen permeabilitywithin the range of 7-30 Barrers, more preferably 8-20 Barrers, and mostpreferably 9-15 Barrers. A Barrer has units of (volume of gas in cm³)(film thickness in cm) (1×10⁻¹⁰)/(time in seconds) (surface area of filmin cm²) (partial pressure of gas in cm of Hg). The film should have acarbon dioxide permeability within the range of 40-80 Barrers. The filmshould have a nitrogen permeability of 10-100 Barrers. The film 10should have a water vapor transmission rate of not more than 20 (gmil/100 in²/day). Preferably the film should have an optical claritywithin the range of about 0.1%-10% as measured by a Hazometer inaccordance with ASTM D1003. The containers must also be capable ofwithstanding radiation sterilization at radiation levels commonly usedin the industry for sterilization.

III. Fabrication of Flexible, Gas Permeable Cell Culture Containers

We turn now to the gas-permeable, flexible cell culture container (20,FIG. 2) formed from the multi-layer films described above. The cellculture container 20 includes a body 21 that is constructed from a firstside wall 22 and an second side wall 24. The side walls 22 and 24 aresealed along their edges to define a containment area 26 for containingthe cell culture media and cells. The side walls 22 and 24 may be sealedby any conventional means such as using heated die and platen which maybe followed by a chill die and platen as is well known in the industry.Also, the side walls 22 and 24 may be sealed using inductive weldingwhich also is known in the industry. For containers constructed fromfilms having as the substrate layer 14 the polymer alloy including thedimer fatty acid polyamide, radio frequency techniques may be used.However, the present invention should not be construed to be limited tousing any one of these fabrication techniques unless otherwise specifiedin the claims.

It is possible to construct various flexible, gas permeable containersfrom the above film in conjunction with other materials.

A. The Non-Adherent Cell Culture Container

FIG. 6 shows a flexible, gas permeable cell culture container 10especially useful for the growth of non-adherent cells. The container 10is constructed from folding and sealing the film shown in FIG. 1 todefine a containment area 30. The first layer of polystyrene faces thecontainment area 30.

B. The Hybrid Cell Culture Bag

FIG. 7 shows a flexible, gas permeable cell culture container 10 whichis suitable for the growth of both adherent and non-adherent cell types.This container is essentially the same as the container set forth inFIG. 6 except the first side wall 22 inner surface 28 is charged with anelectrostatic charge of greater than 40 dynes/cm and preferably about 60dynes/cm. The first side wall 22 with the charge is suitable for growingadherent cells and the second side wall 24 is suitable for growingnon-adherent cells. It is desirable to use some indicia 31 to indicatethe charged side from the uncharged side such as the perimeter geometryof the cell culture container (See FIG. 7a). This would include suchstructural features as a rounding of corners or notching any portion thecontainer, or in any way of varying the shape or structural features ofthe container to indicate the charged side from the uncharged side. Itis also possible to have a raised or embossed area on one side of thecontainer. It is also possible to use color coding or other printedindicia for distinguishing the charged and uncharged sides 22 and 24.

C. Non-Adherent Cell Culture Container with a Clear Panel

FIG. 8 shows a flexible, gas permeable cell culture container having afirst side wall 22 constructed from the film shown in FIG. 1. The secondside wall 24′ is constructed from a film having a substrate 30 ofethylene vinyl acetate having a vinyl acetate content of 18%±2% and aninner layer 32 of HIPS. The first and second side walls 22 and 24′ arebonded along their edges as set forth above or by any suitable method.The second side wall 24′ has an optical clarity as measured by aHazometer in accordance with ASTM D1003 within the range of 0.1%-10%which provides for ease of viewing the cells using a microscope or withthe naked eye. The first side wall 22 would serve as the cell growthsurface for non-adherent cells. It is also possible to apply anelectrostatic charge to the inner surface 28 of the side wall 22 toprovide for a growing surface for adherent cells.

Preferably each of the containers 20 shown in FIGS. 6-8 will includeaccess ports 40 as shown in FIG. 9. The access ports 40 facilitatefilling and emptying of the container 20 of cells or cell culture mediawithout interrupting the closed system. Of course, any number of accessports can be provided as well as a tube set assembly, or the like.

D. Other Containers

It is also desirable to construct containers using the films shown inFIGS. 3-5.

IV. Method of Providing Adjustable Gas Permeability

It is desirable to construct a flexible cell culture container 10 havinga predetermined gas permeability. The predetermined gas permeabilityselected promotes cell growth within the container 10. Preferably, theselected permeability optimizes cell growth.

The gas permeability depends upon the types of polyolefin second layer,the thickness of the individual layers, and the overall thickness of thefilm.

Thus, the method of constructing or fabricating a gas permeable, cellgrowth container having a predetermined gas permeability comprises thefollowing steps: providing a polystyrene, providing an appropriatepolyolefin substrate layer, and coextruding the polystyrene and thepolyolefin producing a layered film having a gas permeability to effectcell growth. Preferably, the cell growth will be optimal.

EXAMPLE 1

A film in accordance with the present invention was coextruded from a0.0003 inch thick layer of polystyrene (K-resin, HIPS) on a 0.0075 inchthick layer of polyolefin alloy (PL-732®). A portion of the film wascorona discharge treated. The film was formed into a flexible containeror bag using a heat seal process. A length of film was cut from a rollof coextruded film. Port fitments, described in the commonly assignedU.S. Pat. No. 4,327,726, which is incorporated herein by reference, wereheat sealed to the film near the midpoint of the length. The film wasfolded across the width of the sheet, near the sealed port fitments. Thefolded sheet with port fitments was placed on a heated brass platen andheat sealed using a heated brass die. The die and platen were operatedat a constant temperature, the die at 280° F., and the platen at 370° F.No chilling dies or devices were employed. After sealing the container,the container was removed from the platen and allowed to air cool. Portclosures were solvent bonded to the port fitments as is known in theart.

The bags were radiation sterilized and employed in cell culture studiesof human progenitor cells. The bags were used to culture progenitorcells in a 10-12 day period in vitro culture. Progenitor cells werederived from purified CD34+cells (stem cells) collected from peripheralblood as mobilized stem cells during a leukopheresis procedure. Thecells were cultured via a process described in co-pending and commonlyassigned patent application Ser. No. 07/855,295 which is incorporatedherein by reference. Coextruded cell culture bags were seded at a celldensity of 0.1-1×10⁵ (cells/ml). Coextruded culture bags were able tosupport an increase in the total number of viable cells during the 10-12day culture period. Typical increases of 40-70 fold were observed. Cellproliferation in excess of 100 fold has been obtained in the culturebags using recombinant growth factors known to support progenitor cellgrowth in vitro. In addition to the increase in cell number, the culturebags were able to support an increase in Colony Forming Cells (“CFC”)and early granulocytes as determined by flow cytometry and cytologicalstaining.

It is understood that, given the above description of the embodiments ofthe invention, various modifications may be made by one skilled in theart. Such modifications are intended to be encompassed by the claimsbelow.

What is claimed is:
 1. A multi-layer, flexible, gas-permeable filmsuitable for forming a cell culture container, the film comprising: afirst layer composed of a polystyrene having a thickness within therange of 0.0001 inches to about 0.0010 inches, the first layer definingan inner cell growth surface; and, a second outer layer adhered to thefirst layer composed of a polymer alloy blend having multiplecomponents, the second layer having a thickness within the range of0.004 inches to about 0.015 inches.
 2. The film of claim 1 wherein thepolystyrene of the first layer is modified by polybutadiene rubber. 3.The film of claim 1 wherein the first layer has a thickness in the rangeof about 0.0002 inches to about 0.0006 inches.
 4. The film of claim 1wherein the first layer has a thickness of about 0.0003 inches.
 5. Thefilm of claim 1 wherein the polymer alloy has three components, a firstcomponent is a styrene-ethylene-butadiene-styrene block copolymer, asecond component is ethylene vinyl acetate, and a third component ispolypropylene.
 6. The film of claim 5 wherein thestyrene-ethylene-butadiene-styrene block copolymer constitutes 40 to 85%by weight of the polymer alloy, the ethylene vinyl acetate constitutes 0to 40% by weight of the polymer alloy, and the polypropylene constitutes10 to 40% by weight of the polymer alloy.
 7. The film of claim 1 whereinthe polymer alloy has four components.
 8. The film of claim 7 whereinthe four component polymer alloy has a first component of apolypropylene, a second component selected from the group consistingessentially of an ultra low density polyethylene and polybutene-1, athird component of a dimer fatty acid polyamide, and a fourth componentof a styrene-ethylene-butadiene-styrene block copolymer.
 9. The film ofclaim 8 wherein the first component constitutes within the range of30-60% by weight of the polymer alloy, the second component constituteswithin the range of 25%-50% by weight of the polymer alloy, the thirdcomponent constitutes within the range of 5%-40% by weight of thepolymer alloy, and the fourth component constitutes 5%-40% by weight ofthe polymer alloy.
 10. The film of claim 1 further comprising a tielayer interposed between the first and second layers, the tie layerproviding adhesive compatibility between the first and second layers.11. The film of claim 10 wherein the tie layer is composed of a gaspermeable olefin.
 12. The film of claim 11 wherein the gas permeableolefin is an ethylene polymer containing vinyl acetate within the rangeof 16%-32% by weight.
 13. The film of claim 12 wherein the ethylenevinyl acetate has a vinyl acetate content of 28% by weight.
 14. The filmof claim 1 further comprising a skin layer adhered to the second layeropposite the first layer, the skin layer providing additional strengthand scratch resistance.
 15. The film of claim 14 wherein the skin layeris composed of polypropylene.
 16. The film of claim 14 wherein the skinlayer is composed of polyethylene.
 17. The film of claim 14 wherein theskin layer has a thickness within the range of 0.0001 inches to about0.002 inches.
 18. The film of claim 17 wherein the skin layer has athickness of about 0.0005 inches.
 19. The film of claim 1 wherein thefirst layer has an electrostatic charge greater than 40 dynes/cm.
 20. Amulti-layer, flexible, gas-permeable film suitable for forming a cellculture container, the film comprising: a first layer composed of apolystyrene having a thickness within the range of 0.0001 inches toabout 0.0010 inches, the first layer defining a cell growth surface; asecond layer adhered to the first layer composed of a polymer alloyblend having multiple components; and, wherein the film having physicalproperties within the range: a>10,000 but <30,000 b>9 but <15 c>40 but<80 d>10 but <100 e<20 wherein: a is the flexural modulus in psi of thefilm measured according to ASTM D-790; b is the oxygen permeability inBarrers; c is the carbon dioxide permeability in Barrers; d is thenitrogen permeability in Barrers; and e is the water vapor transmissionrate in (g mil/100 in ²/day).
 21. The film of claim 20 wherein the filmhas an optical clarity within the range of 0.1%-10% as measured by aHazometer under ASTM D1003.
 22. The film of claim 20 wherein the polymeralloy has three components, a first component isstyrene-ethylene-butadiene-styrene block copolymer, a second componentis ethylene vinyl acetate, and a third component is polypropylene. 23.The film of claim 22 wherein the styrene-ethylene-butadiene-styreneblock copolymer constitutes 40%-85% by weight of the polymer alloy, theethylene vinyl acetate constitutes 0 to 40%% by weight of the polymeralloy, and the polypropylene constitutes 10 to 40% by weight of thepolymer.
 24. The film of claim 20 wherein the second layer is composedof a four component polymer alloy.
 25. The film of claim 24 wherein thefour component polymer alloy has a first component of a polypropylene, asecond component selected from the group consisting essentially of anultra low density polyethylene and polybutene-1, a third component of adimer fatty acid polyamide, and a fourth component of astyrene-ethylene-butadiene-styrene block copolymer.
 26. The film ofclaim 25 wherein the first component constitutes within the range of30-60% by weight of the polymer alloy, the second component constituteswithin the range of 25%-50% by weight of the polymer alloy, the thirdcomponent constitutes within the range of 5%-40% by weight of thepolymer alloy and the fourth component constitutes 5%-40% by weight ofthe polymer alloy.
 27. A cell culture container comprising a first andsecond side wall each having an interior surface that cooperate todefine a containment area, wherein at least the first side wallscomposed of a film of any of claims 1-26.
 28. The container of claim 27wherein the polystyrene first layer faces the containment area.
 29. Aflexible, gas-permeable cell culture container suitable for culturingcells, the container comprising: a first and second side wall eachhaving edges, the first and second side walls being sealed together attheir respective side wall edges to provide a containment area, whereinat least the first side wall is composed of a first layer of polystyrenehaving a thickness within the range of 0.0001 inches to a out 0.0010inches the first layer facing an interior of the container to define acell growth surface, and, a second layer adhered to the first layer of apolyolefin, the second layer having thickness within the range of 0.004inches to about 0.015 inches.
 30. The cell culture container of claim 29further comprising at least one access port for accessing thecontainment area.
 31. A method for fabricating a multi-layered filmsuitable for forming a container for culturing cells comprising thesteps of: providing a polystyrene cell growth surface; providing apolymer alloy blend having multiple components; and, coextruding thepolystyrene and the polymer alloy blend producing a layered film havinga gas permeability to promote cell growth.
 32. The method of claim 31wherein the polystyrene layer has a thickness within the range of about0.0001 to about 0.0010 inches, and wherein the polyolefin layer has athickness within the range of about 0.004 to about 0.015 inches.
 33. Aflexible, gas-permeable cell culture container suitable for culturingcells, the container comprising: a first side wall of the containerbeing suitable for growing adherent cells, the first side wall comprisesa first layer of polystyrene having a thickness within the range of0.0001 inches to about 0.0010 inches, and, a second layer adhered to thefirst layer of a polymer alloy having multiple components, the secondlayer having a thickness within the range of 0.004 inches to about 0.015inches; a second side wall attached to the first side wall for growingnon-adherent cells; and, means associated with the container fordistinguishing the first side wall from the second side wall.
 34. Thecontainer of claim 33 wherein the means for distinguishing the firstside wall from the second side wall is the geometry of the container.35. A method for culturing cells comprising the steps of: providing aflexible cell culture container having at least one side wall of a filmhaving a first layer of a polystyrene having a thickness from 0.0001inches to about 0.0010 inches, and a second layer adhered to the firstlayer of a polymeric material having a thickness from 0.004 inches toabout 0.015 inches, the first layer faces an interior of the containerto provide a cell growth surface; adding to the container a cell growthmedium; and seeding the container with cells to be grown.
 36. The methodof claim 35 wherein the second layer is a polymer alloy having multiplecomponents.
 37. The film of claim 36 wherein the polymer alloy has threecomponents, a first component is a styrene-ethylene-butadiene-styreneblock copolymer, a second component is ethylene vinyl acetate, and athird component is polypropylene.
 38. The film of claim 37 wherein thestyrene-ethylene-butadiene-styrene block copolymer constitutes 40 to 85%by weight of the polymer alloy, the ethylene vinyl acetate constitutes 0to 40% by weight of the polymer alloy, and the polypropylene constitutes0 to 40% by weight of the polymer alloy.
 39. The film of claim 36wherein the second layer is composed of a four component polymer alloy.40. The film of claim 39 wherein the four component polymer alloy has afirst component of a polypropylene, a second component selected from thegroup consisting of an ultra low density polyethylene and polybutene-1,a third component of a dimmer fatty acid polyamide, and a fourthcomponent of a styrene-ethylene-butadiene-styrene block copolymer. 41.The film of claim 40 wherein the first component constitutes within therange of 30-60% by weight of the polymer alloy, the second componentconstitutes within the range of 25%-50% by weight of the polymer alloy,the third component constitutes within the range of 5%-40% by weight ofthe polymer alloy, and the fourth component constitutes 5%-40% by weightof the polymer alloy.
 42. The film of claim 35 further comprising a tielayer interposed between the first and second layers, the tie layerproviding adhesive compatibility between the first and second layers.